Benjamin T. Wall, Francis B. Stephens, Kanagaraj Marimuthu, Dumitru Constantin-Teodosiu, Ian A. Macdonald, and Paul L. Greenhaff

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1 J Appl Physiol 112: , First published November 3, 2011; doi: /japplphysiol Acute pantothenic acid and cysteine supplementation does not affect muscle coenzyme A content, fuel selection, or exercise performance in healthy humans Benjamin T. Wall, Francis B. Stephens, Kanagaraj Marimuthu, Dumitru Constantin-Teodosiu, Ian A. Macdonald, and Paul L. Greenhaff School of Biomedical Sciences, University of Nottingham Medical School, Queen s Medical Centre, Nottingham, United Kingdom Submitted 30 June 2011; accepted in final form 28 October 2011 Wall BT, Stephens FB, Marimuthu K, Constantin-Teodosiu D, Macdonald IA, Greenhaff PL. Acute pantothenic acid and cysteine supplementation does not affect muscle coenzyme A content, fuel selection, or exercise performance in healthy humans. J Appl Physiol 112: , First published November 3, 2011; doi: /japplphysiol Reduced skeletal muscle free coenzyme A (CoASH) availability may decrease the contribution of fat oxidation to ATP production during high-intensity, submaximal exercise or, alternatively, limit pyruvate dehydrogenase complex (PDC) flux and thereby carbohydrate oxidation. Here we attempted to increase the muscle CoASH pool in humans, via pantothenic acid and cysteine feeding, in order to elucidate the role of CoASH availability on muscle fuel metabolism during exercise. On three occasions, eight healthy male volunteers (age yr, body mass index kg/m 2 ) cycled at 75% maximal oxygen uptake (V O 2max ) to exhaustion, followed by a 15-min work output performance test. Muscle biopsies were obtained at rest, and after 60 min and min of exercise (time to exhaustion on baseline visit) on each occasion. Two weeks following the first visit (baseline), 1 wk of oral supplementation with either 3 g/day of a placebo control (glucose polymer; CON) or 1.5 g/day each of D-pantothenic acid and L-cysteine (CP) was carried out prior to the second and third visits in a randomized, counterbalanced, double-blind manner, leaving a 3-wk gap in total between each visit. Resting muscle CoASH content was not altered by supplementation in any visit. Following 60 min of exercise, muscle CoASH content was reduced by 13% from rest in all three visits (P 0.05), and similar changes in the respiratory exchange ratio, glycogenolysis ( 235 mmol/kg dry muscle), PCr degradation ( 57 mmol/kg dry muscle), and lactate ( 25 mmol/kg dry muscle) and acetylcarnitine ( 12 mmol. kg/dry muscle) accumulation was observed during exercise when comparing visits. Furthermore, no difference in work output was observed when comparing CON and CP. Acute feeding with pantothenic acid and cysteine does not alter muscle CoASH content and consequently does not impact on muscle fuel metabolism or performance during exercise in humans. coenzyme A; fuel utilization; skeletal muscle; exercise Address for reprint requests and other correspondence: B. T Wall, School of Biomedical Sciences, Univ. of Nottingham Medical School, Queen s Medical Centre, Nottingham NG7 2UH, United Kingdom ( COENZYME A (CoASH) fulfills several distinct roles in muscle energy metabolism. First, as a substrate for the enzyme acyl- CoA synthetase (ACS), CoASH allows the activation of cytosolic fatty acids to fatty acyl-coas before their subsequent delivery to the mitochondria via the carnitine shuttle system (3, 19). Second, in the final stage of the carnitine shuttle system, mitochondrial CoASH is required for the carnitine palmitoyltransferase 2 (CPT2)-mediated transesterification of acylcarnitine to carnitine and acyl-coa. Third, a viable supply of free CoASH is necessary for the final stage of mitochondrial fatty acid -oxidation, where -ketoacyl-coa is sequentially cleaved by the thiol group of another molecule of CoASH to form acetyl-coa and acyl-coa (12). Fourth, the pyruvate dehydrogenase complex (PDC)-mediated oxidative decarboxylation of pyruvate to acetyl-coa also requires an available pool of mitochondrial free CoASH (8, 12). Finally, free CoASH is also a key substrate for -ketoglutarate dehydrogenase within the tricarboxylic acid (TCA) cycle and is therefore necessary for TCA flux (12). Despite these well-documented roles, the influence of muscle CoASH availability on skeletal muscle fuel metabolism during exercise in humans remains to be fully characterized. It has been proposed that a reduced availability of muscle free CoASH to the cytosolic ACS reaction may limit fat oxidation during exercise (25 27). Indeed, a significant reduction in muscle free CoASH availability has consistently been reported during exercise 70% maximal oxygen uptake (V O 2max ) (but not at lower intensities) where fat oxidation would be expected to be low comparative to lower exercise intensities, and this is entirely accounted for by a parallel accumulation of acetyl-coa (6, 7, 20, 29). However, the subcellular localization of this decline in CoASH availability during high-intensity, submaximal exercise remains unknown. If mitochondrial CoASH becomes depleted, for example, this could reduce fat oxidation by virtue of CoASH s role in the CPT2 reaction or within fatty acid -oxidation or, alternatively, it could limit PDC flux due to the dependence of the reaction on free CoASH availability. Indeed, carnitine is thought to maintain a viable pool of free CoASH by buffering acetyl groups to acetylcarnitine thereby allowing continued PDC flux (30), and we have recently reported that increasing muscle carnitine availability augmented PDC activation and flux during exercise at 80% V O 2max (36). The aim of the present study therefore was to elevate the muscle free CoASH pool in healthy humans via 7 days of oral supplementation with pantothenic acid and cysteine (precursors of endogenous CoASH biosynthesis) in order to further elucidate the role of CoASH availability in the regulation of muscle fuel metabolism during exercise. Pantothenic acid is the primary substrate for the pantothenate kinase (PK; the rate-limiting step in CoASH biosynthesis) reaction and, assuming adequate cysteine is available from the diet, it has been suggested that dietary pantothenic acid may regulate CoASH biosynthesis (22, 23). We hypothesized that if CoASH avail /12 Copyright 2012 the American Physiological Society

2 ability was limiting to fat oxidation, then elevating CoASH would reduce the respiratory exchange ratio (RER) and spare muscle glycogen utilization during steady-state exercise at 75% V O 2max. On the other hand, if CoASH availability was limiting to PDC flux, we hypothesized that increasing muscle CoASH availability would result in a better matching of glycolytic and TCA flux, i.e., increased acetyl-coa and acetylcarnitine formation, resulting in reduced muscle phosphocreatine hydrolysis and lactate accumulation for the same glycogen utilization. In keeping with both these hypotheses, 1 wk of dietary pantothenic acid supplementation (1.5 g/day) has previously been shown to reduce blood lactate concentration compared with control during steady-state exercise at 75% V O 2max (16); however, the underlying mechanism within skeletal muscle was not investigated. Finally, we hypothesized that if pantothenic acid and cysteine supplementation could impact on muscle CoASH availability and muscle fuel metabolism during exercise, irrespective of the mechanism by which the latter was achieved, it could reasonably be expected to increase work output during a subsequent exercise performance test. METHODS Human volunteers. Eight healthy, nonsmoking, nonvegetarian, recreationally active males (age yr; body mass kg; body mass index kg/m 2 ; V O 2max ml kg 1 min 1 ) participated in this study. The study was approved by the University of Nottingham Medical School Ethics Committee in accordance with the Declaration of Helsinki. Prior to the study, each subject completed a routine medical screening and a general health questionnaire to ensure their suitability to take part. All gave their written consent to participate in the study and were aware that they were free to withdraw from the experiment at any time. Pretesting. Fourteen days before the trial, each subject s V O 2max was measured using an online gas analyzer (Vmax; SensorMedics, Anaheim, CA) during a continuous and incremental, exhaustive exercise protocol on an electronically braked cycle ergometer (Lode NV Instrumenten, Groningen, the Netherlands). Achievement of V O 2max was accepted when a plateau in oxygen consumption occurred despite a further increase in workload, which was confirmed during a repeat test 3 days later. Thereafter and at least 1 wk prior to the first experimental visit, subjects were familiarized to the experimental exercise protocol during which the workload required to elicit 75% of V O 2max was also confirmed. Experimental protocol. Volunteers reported to the laboratory at 0830 on three occasions over a 6-wk period, each visit being separated by 3 wk. The protocol for each of these visits is depicted in Fig. 1. Subjects arrived after an overnight fast having abstained from strenuous exercise and alcohol consumption for at least 48 h, and caffeine for at least 24 h. On arrival at the laboratory on each visit, subjects were weighed, voided their bladder, and then rested in a semisupine MUSCLE COENZYME A AND FUEL SELECTION 273 position while a cannula was inserted into an antecubital vein in the nondominant arm for subsequent venous blood collections. Volunteers then underwent a glycogen-depleting exercise protocol consisting of continuous bicycling exercise for 60 min at 75% of V O 2max followed by intermittent exercise at the same intensity until volitional exhaustion. We have previously shown that this protocol is effective at reducing mixed fiber muscle glycogen content to 80 mmol/kg dry muscle (4). Exhaustion was defined as the inability to maintain 70 rpm on the cycle ergometer for 1 min immediately following a 5-min rest period. The time taken to reach exhaustion was recorded for the first visit (totaling min of exercise) and repeated for the two subsequent visits with the timing of rest periods kept identical. At the point of exhaustion, subjects were permitted a 5-min rest after which they performed a 15-min work output (kj) performance test. This all-out performance test involved using the ergometer linear mode function, where work output is dependent on volitional cycling cadence. This performance test has been shown to be a more reliable measurement of endurance exercise performance than other tests such as cycling at a fixed exercise workload to volitional exhaustion (15) and has been used previously in our laboratory as it appears to be sensitive to skeletal muscle glycogen availability and/or PDC flux capacity (31, 36). Supplementation protocol. After the first experimental visit, a 2-wk rest/washout period was allocated, at which point volunteers began 1 wk of oral supplementation with either 1.5 g/day each of L-cysteine and D-pantothenic acid (CP; Holland and Barrett, Warwickshire, UK), or 3 g/day of a glucose polymer control (CON; Maxijul, UK). Following the second experimental visit and a further 2-wk rest/ washout period volunteers repeated the supplementation protocol. The order of the supplementation was randomized and counterbalanced in a double-blind manner. A 2-wk recovery/washout period was selected to prevent any effect of glycogen supercompensation following the exercise visit and because it has previously been shown that 80% of an oral pantothenic acid dose is eliminated from the body within 5 days (32). Sample collection and analysis. On each experimental study day, venous blood samples were collected at rest while subjects rested in a semisupine position, and every 10 min during the first 60 min of continuous exercise at 75% V O 2max, and at the point of exhaustion. Following collection, blood glucose and lactate concentration was determined immediately using an autoanalyzer (YSI 2300 STATplus, Yellow Springs Instruments, Yellow Springs, OH). Expired CO 2 and O 2 were measured for 3 min at the end of every 10 min of exercise over the first 60 min of exercise at 75% V O 2max using an online gas analyzer (Vmax; SensorMedics, Anaheim, CA) and the values recorded were used to calculate the respiratory exchange ratio (RER). On each experimental visit, muscle biopsy samples were obtained from the vastus lateralis muscle at rest, within5softheendof60min of exercise at 75% V O 2max (while subjects were seated on the cycle ergometer) and at the point of exhaustion using the percutaneous needle biopsy technique (2). Muscle samples were snap frozen in liquid nitrogen immediately after removal from the limb and were Fig. 1. Experimental protocol.v O 2max, maximal oxygen uptake.

3 274 MUSCLE COENZYME A AND FUEL SELECTION Table 1. Skeletal muscle metabolites at rest and following cycling exercise at 75 % V O 2max continuously for 60 min and intermittently to the point of exhaustion, before (Baseline), after 1 wk of orally ingesting a glucose polymer (3 g/day; CON), and after 1 wk of orally ingesting pantothenic acid and cysteine (1.5 g of each per day; CP) Baseline CON CP Rest PCr Glycogen Lactate Acetyl-CoA Acetylcarnitine CoASH min PCr Glycogen Lactate Acetyl-CoA * * * Acetylcarnitine CoASH * Exhaustion PCr Glycogen Lactate Acetyl-CoA * * * Acetylcarnitine * * CoASH All values are means standard error of the mean (SE) and expressed as mmol/kg dry muscle with the exception of acetyl-coa and CoASH which are expressed as mol/kg dry muscle. Significantly different from corresponding resting value: *P 0.05, P Significantly different from corresponding baseline value: P 0.05, P Skeletal muscle free CoASH content. The skeletal muscle free CoASH content at rest, following 60 min continuous exercise at 75% V O 2max, and following intermittent exercise to exhaustion in the baseline, CON, and CP visits is displayed in Table 1. At rest, there were no differences between visits in muscle free CoASH content. Muscle free CoASH content showed a significant exercise effect (P 0.05), i.e., it declined from rest by 13% over 60 min of exercise. However, there was no difference between visits in the magnitude of the decline. Furthermore, muscle free CoASH content was unchanged at exhaustion compared with the 60-min time point in all visits. RERs. The RER over the first 60 min of exercise at 75% V O 2max is presented in Fig. 2A. As expected, there was a significant time effect (P 0.001), but there was no difference between visits. Skeletal muscle metabolites. Muscle metabolite concentrations at rest, following 60 min exercise, and at exhaustion during baseline, CON, and CP visits are shown in Table 1. At rest, there were no differences between visits. Sixty minutes of exercise depleted muscle glycogen (P 0.001) and PCr (P 0.001) content to the same extent during all three visits (Figs. 2B and 3A, respectively), and also increased muscle lactate (P 0.001), acetyl-coa (P 0.05), and acetylcarnitine (P 0.001) (Fig. 3, B D, respectively), with the magnitude of increase being lower in the CON and CP visits compared with the baseline visit. There were no differences in muscle metabolites when comparing the CON and CP visits. At the point of exhaustion, muscle glycogen was depleted to 13% of the resting content (P 0.001), but there was no difference in the magnitude of depletion between visits. Muscle acetylcarnitine and acetyl-coa content remained elevated above resting values at the point of exhaustion, fourfold (P 0.05) and twofold (P 0.05) fold, respectively, but muscle lactate and PCr content had returned to resting levels. More- then freeze dried and stored at 80 C. Freeze dried muscle was dissected free of all visible blood and connective tissue, powdered, and used for the determination of muscle acetylcarnitine, acetyl-coa, and CoASH using the radioenzymatic methods described previously by Cederblad et al. (5), and muscle phosphocreatine (PCr), lactate, and glycogen using the spectrophotometric methods of Harris et al. (13). Muscle glycogen and PCr utilization, and lactate and acetylcarnitine accumulation over the initial 60 min of exercise was calculated as the difference between rest and the 60-min biopsy sample. This was not done for the exhaustion time point because of the variation between individuals in exercise time to exhaustion. Statistical analysis. A two-way ANOVA was performed to detect differences between time (rest, 60 min of exercise, and exhaustion) and treatment (baseline, CON, and CP). When a significant time or treatment effect was observed, a Bonferonni post hoc test was performed to locate individual differences. Statistical significance was declared at P All the values presented in text, tables, and figures represent means standard error of the mean (SE). RESULTS Fig. 2. Respiratory exchange ratios (RER; A) and skeletal muscle glycogen utilization (B) during 60 min of exercise at 75% V O 2max before (baseline), after 1 wk of orally ingesting a glucose polymer (3 g/day; CON), and after 1 wk of orally ingesting pantothenic acid and cysteine (1.5 g of each per day; CP). All values are means standard error of the mean (SE).

4 MUSCLE COENZYME A AND FUEL SELECTION 275 Fig. 3. Skeletal muscle phosphocreatine (PCr) degradation (A), lactate accumulation (B), acetyl-coa accumulation (C), and acetylcarnitine accumulation (D) during 60 min of exercise at 75% V O 2max before (baseline), after 1 wk of orally ingesting a glucose polymer (3 g/day; CON), and after 1 wk of orally ingesting pantothenic acid and cysteine (1.5 g of each per day; CP). All values are means SE. Significantly different from corresponding baseline (0): P 0.05, P over, at the point of exhaustion there was no difference between visits in any of the measured metabolites. Blood glucose and lactate concentrations. The concentration of glucose and lactate in whole blood at rest, at 10-min intervals throughout the first 60 min of exercise, and at the point of exhaustion is presented in Fig. 4. Blood glucose concentration was maintained at 4.3 mmol/l throughout the first 60 min of exercise and declined to 3.5 mmol/l at the point of exhaustion in all visits, with no differences detected between visits (Fig. 4A). In all visits, blood lactate concentration increased from rest to 3.7 mmol/l during exercise, then declined to 2 mmol/l at the point of exhaustion, again, with no differences apparent between visits (Fig. 4B). Exercise performance. Work output (kj) achieved in the exercise performance test performed directly after exhaustive exercise is presented in Fig. 5. Work output was greater than baseline in CON (24%; P 0.01) and CP (15%; P 0.05). However, no significant difference was detected between CON and CP. DISCUSSION The principal finding of the present study was that 7 days of oral feeding with D-pantothenic acid and L-cysteine (1.5 g/day each) did not increase muscle coenzyme A (CoASH) availability at rest or during exercise and did not influence muscle fuel metabolism during steady-state exercise at an intensity where muscle CoASH availability is reduced compared with rest. Furthermore, supplementation did not lead to an improvement in exercise performance compared with a placebo control visit. While the effect of pantothenic acid feeding on exercise performance has been examined previously (37), an explicit attempt to elevate skeletal muscle free CoASH in humans via feeding with its biological precursors (pantothenic acid and cysteine) has, to the authors knowledge, not been attempted before. The ingestion of CoASH per se results in its prompt degradation to its precursors within the gut, whereas dietary pantothenic acid is readily absorbed primarily via a saturable, sodium-dependent active transport process (28). Accordingly, we chose the present approach of feeding CoASH precursors. The phosphorylation of pantothenic acid to 4 -phosphopantothenic acid by pantothenate kinase (PK) is regarded as the rate-limiting step in muscle CoASH synthesis (1) and has been suggested to be regulated, in part, by dietary availability of pantothenic acid in humans (23). Cysteine, on the other hand, is suggested to become limiting to CoASH synthesis only in the unlikely event of a deficiency. Thus we employed pantothenic acid supplementation in an attempt to drive CoASH formation and cysteine ingestion simply to be sure no defi-

5 276 MUSCLE COENZYME A AND FUEL SELECTION Fig. 4. Blood glucose (A) and lactate (B) concentrations during 60 min of exercise at 75% V O 2max before (baseline), after 1 wk of orally ingesting a glucose polymer (3 g/day; CON) and after 1 wk of orally ingesting pantothenic acid and cysteine (1.5 g of each per day; CP). All values are means SE. ciency existed. The normal plasma concentration of pantothenic acid in healthy humans is 7 mol/l (9), which is below the reported 11 mol/l K m for the transport of pantothenic acid into muscle (18, 32) and suggests that pantothenic acid transport into muscle is not habitually saturated. Indeed, in isolated heart preparations, the excess provision of pantothenic acid and cysteine has been shown to result in acute elevations of CoASH content by % (17, 22, 24). Given the bioavailability of ingested pantothenic acid is 40 60% and the daily urinary excretion rate is 60% of oral intake (32, 34), and assuming a volume of distribution similar to that of the volume of extracellular fluid (33), it would be expected that each 500 mg (i.e., 3 times per day) dose of pantothenic acid would have elevated plasma concentrations at least threefold, and thus far above the necessary threshold for intramuscular transport. Accordingly, assuming a complete conversion of intramuscular pantothenic acid to CoASH, this would be expected to result in at least a doubling of muscle CoASH content, which should have been easily detectable. Thus it is not immediately obvious why we did not observe an increase in muscle CoASH content in the present study, particularly as it has been previously demonstrated that 1 wk of feeding with the same dose of pantothenic acid used in the present study results in a reduced blood lactate concentration during exercise at 75% V O 2max compared with a placebo control (16). A possible explanation lies in the kinetics of the PK reaction. Specifically, PK is inhibited in vitro by several energy substrates and hormones including glucose, pyruvate, -hydroxybutyrate and fatty acids (21) as well as by means of end-product inhibition by CoASH and acetyl-coa (10, 11). Thus the in vivo situation represents a much more complex physiological milieu, which may exert tighter control over the PK reaction compared with the controlled in vitro experiments performed predominantly on cardiac tissue where increases in CoASH have previously been observed (17, 22, 24). It has previously been hypothesized that the availability of cytosolic CoASH to acyl-coa synthetase (ACS) may exert a degree of control over fat oxidation during exercise by controlling the rate of activation and subsequent mitochondrial delivery of fatty acids (25 27). In support, it has consistently been reported that exercise above 70% V O 2max, where fat oxidation would be expected to be less than at lower intensities (35), results in reduced muscle CoASH availability, whereas lighter exercise does not disturb the CoASH pool (6, 7, 20, 29). In agreement with the literature, resting skeletal muscle free CoASH concentration in the present study was 15 mol/l intracellular water (6, 7). While subcellular fractions of CoASH have not been determined in skeletal muscle, analyses of cardiac tissue have shown that only 10% of the total cellular CoASH pool exists within the cytosol (14, 19). If these data from cardiac muscle are representative of skeletal muscle, it is likely that skeletal muscle cytosolic CoASH content at rest is below that of its reported in vitro K m for ACS (7 mol/l) (19), and that any further reduction in CoASH availability during exercise would clearly limit the reaction. In agreement with the literature, muscle CoASH was reduced by 13% following 60 min of exercise in the present study (and was partly accounted for by an increase in acetyl-coa), and therefore its availability could have been a potential limitation to fat oxidation, as discussed above and previously by others (25 27). The reported decline in CoASH availability during exercise may be cytosolic or mitochondrial. If the latter were the case, we hypothesized that this could conceivably limit fat oxidation or may actually exert a degree of inhibition on PDC flux. In Fig. 5. Work output generated during a 15 min all-out exercise performance test performed immediately following exhaustive exercise at 75% V O 2max before (baseline), after 1 wk of orally ingesting a glucose polymer (3 g/day; CON), and after 1 wk of orally ingesting pantothenic acid and cysteine (1.5 g of each per day; CP). All values are means SE. Significantly different from corresponding baseline (0): P 0.05.

6 keeping with this, we have recently reported that a carnitinemediated improvement in acetyl-coa buffering during exercise at 80% V O 2max (and, presumably increased free muscle CoASH availability) led to a greater PDC activation status and flux and, as such, better matching of glycolytic and TCA cycle flux in healthy volunteers (36). Moreover, the availability of muscle carnitine and free CoASH are closely associated in exercising skeletal muscle (6, 7). In the present study, we therefore aimed to determine whether 7 days of feeding with the precursors of endogenous CoASH biosynthesis (i.e. pantothenic acid and cysteine) would either augment fat oxidation or result in a better matching of glycolytic and TCA cycle flux. With respect to the former, we directly assessed muscle glycogen utilization during 60 min of exercise at 75% V O 2max as well as measuring respiratory exchange ratios (RER) during exercise as an indicator of muscle fuel selection. Given that we observed no differences between our CON and CP visits in either of these parameters, it is clear that no such shift in muscle fuel selection occurred. Regarding the latter, we demonstrated that PDC flux, as indicated through muscle acetyl- CoA and acetylcarnitine accumulation, was not altered between CON and CP visits, nor did anaerobic glycolytic flux (as evident by the rate of lactate accumulation) differ between visits, demonstrating no change in the matching of glycolytic and TCA flux. Furthermore, in view of the absence of an effect of the treatment on exercise fuel metabolism, it is perhaps unsurprising that we failed to observe an effect on exercise performance. Taken together, the reasons underpinning our absence of effect are undoubtedly due to the failure of our supplementation protocol to alter muscle free CoASH content. Indeed, even the sum of muscle contents of free CoASH and acetyl-coa was identical between visits. This underlines the importance of developing strategies capable of manipulating the muscle CoASH pool such that its role in the regulation of muscle fuel selection can be realized. It is important to note that an apparent order effect influenced the baseline visit in the present study. That is to say, when comparing either the CON or CP visit to baseline, the accumulation of muscle lactate, acetyl-coa, and acetylcarnitine during exercise was reduced, while exercise performance was better. This is most likely explained by an order effect, in that the baseline visit was always conducted first with the subsequent visits occurring in a randomized order. This order effect did not influence the comparison of CON with CP visits and was probably attributable to a training response occurring due to subjects undertaking an average of 1.5 training visits prior to commencement of supplementation. In summary, the present study demonstrates that 7 days of oral supplementation with pantothenic acid and cysteine (1.5 g/day each) in healthy volunteers does not result in an increase in the muscle free CoASH availability and does not influence muscle fuel selection during exercise at 75% V O 2max or enhance exercise performance. ACKNOWLEDGMENTS We thank QinetiQ for their sponsorship of this research. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). MUSCLE COENZYME A AND FUEL SELECTION AUTHOR CONTRIBUTIONS Experiments in the manuscript were conducted in the School of Biomedical Sciences, University of Nottingham. All authors approved the final version of the manuscript to be published and all authors contributed to drafting the article and revising it critically for important intellectual content. All authors contributed to the conception and design, or analysis and interpretation of the data in the manuscript. 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